Role of TRPC6 in kidney damage after acute ischemic kidney injury

Transient receptor potential channel subfamily C, member 6 (TRPC6), a non-selective cation channel that controls influx of Ca2+ and other monovalent cations into cells, is widely expressed in the kidney. TRPC6 gene variations have been linked to chronic kidney disease but its role in acute kidney injury (AKI) is unknown. Here we aimed to investigate the putative role of TRPC6 channels in AKI. We used Trpc6−/− mice and pharmacological blockade (SH045 and BI-749327), to evaluate short-term AKI outcomes. Here, we demonstrate that neither Trpc6 deficiency nor pharmacological inhibition of TRPC6 influences the short-term outcomes of AKI. Serum markers, renal expression of epithelial damage markers, tubular injury, and renal inflammatory response assessed by the histological analysis were similar in wild-type mice compared to Trpc6−/− mice as well as in vehicle-treated versus SH045- or BI-749327-treated mice. In addition, we also found no effect of TRPC6 modulation on renal arterial myogenic tone by using blockers to perfuse isolated kidneys. Therefore, we conclude that TRPC6 does not play a role in the acute phase of AKI. Our results may have clinical implications for safety and health of humans with TRPC6 gene variations, with respect to mutated TRPC6 channels in the response of the kidney to acute ischemic stimuli.

Transient receptor potential (TRP) channels are a group of ion channels located mostly on the plasma membrane of numerous cell types with a relatively large non-selective permeability to cations 1,2 . Mammalian TRP channel family comprises 28 members, which share some structural similarity with each other 2,3 . Transient receptor potential canonical or classical 6 (TRPC6) are non-selective Ca 2+ permeable cation channels expressed in renal tissue including glomerular podocytes, mesangial cells, endothelial cells, tubulointerstitial vascular and epithelial cells, as well as in renal blood vessels 4 . Ca 2+ influx through TRPC6 maintains the integrity of glomerular filtration barrier by interacting with nephrin, podocin, CD2-associated protein, and α-actinin-4 directly or indirectly 5 . Mutations in TRPC6 lead to familial forms of focal segmental glomerulosclerosis (FSGS) and to end stage kidney disease 6,7 . Of note, TRPC6 dysregulation is also linked to progression of acquired forms of proteinuric kidney disease 8,9 . As a result of the TRPC6 activation, intracellular Ca 2+ concentration in the podocyte increases and ultimately causes programmed cell death leading to progressive kidney failure. Recent evidence strongly suggests that TRPC6 also contributes to renal fibrosis and immune cell infiltration in the unilateral ureteral obstruction mouse model of progressive renal interstitial fibrosis 9,10 .
Initiation of renal fibrosis is often caused by acute kidney injury (AKI), an increasingly common complication occurring in critically ill patients with high morbidity and mortality 11  www.nature.com/scientificreports/ primarily linked to the severity of tubular damage. In fact, most theories consider renal tubular cells as the main culprit in AKI. Ischemia/reperfusion injury (IRI)-induced epithelial damage-particularly in the outer medulla-, tubular obstruction, Ca 2+ overload, loss of cytoskeletal integrity, and loss of cell-matrix adhesion are main consequences of ischemia and initiators of subsequent fibrosis 12,13 . Recent data show that in vivo TRPC6 inhibition by BI-749327 ameliorates renal fibrosis in the unilateral obstructive nephropathy (UUO) model 14 , where the primary feature is tubular injury as a result of obstructed urine flow. Ischemia is frequently involved in AKI and correlates with oxidative stress and inflammation 15,16 . Reactive oxygen species (ROS) produced by NADPH oxidases (NOX) contribute to activation of TRPC6 channels 17,18 . Moreover, TRPC6 is emerging as a functional element to control calcium currents in immune cells, thereby regulating transendothelial migration, chemotaxis, phagocytosis, and cytokine release 19,20 . In a recent study, Shen et al. found that silencing TRPC6 could prevent necroptosis of renal tubular epithelial cells upon IRI 21 . These findings indicate that inhibition of TRPC6 may protect the kidney from IRI making it a promising target to ameliorate AKI. This is of special interest and of potential clinical relevance as novel chemical substances selectively acting on TRPC6 have been recently developed for in vivo experiments. In particular, the diterpene (+)-larixol and its derivative SH045, as well as BI-749327 which is another known TRPC6 blocker, have been developed for selective inhibition of TRPC6 and translational treatment of TRPC6 channelopathies 14,22,23 .
To the best of our knowledge, no studies investigated the potential role of TRPC6 channels in AKI. Therefore, we tested the hypothesis that TRPC6 inhibition is renoprotective in AKI. We induced AKI by ischemia reperfusion injury in wild-type (WT) and Trpc6 −/− mice. Furthermore, we used pharmacological blockers (SH045 and BI-749327) in vivo and ex vivo to verify findings from the genetical model and to evaluate effects of TRPC6 inhibition in IRI-induced AKI and intrarenal regulation of blood flow. Serum markers, renal damage marker expression, histological analysis of renal tissue damage and cellular infiltration were performed. We conclude that neither lacking nor pharmacological inhibition of TRPC6 ameliorates short-term outcomes of AKI.

Results
The impact of Trpc6 deficiency on renal damage after renal IRI. To examine a possible role of Trpc6 deficiency in AKI, we performed comparative in vivo studies using Trpc6 −/− and WT mice (Fig. 1A). Twenty-four hours after IRI WT mice showed similar serum creatinine level compared to Trpc6 −/− mice (P = 0.18; Fig. 1B). Renal expression of tubular damage marker neutrophil gelatinase-associated lipocalin (Ngal) and kidney injury molecule 1 (Kim1) showed no difference between WT and Trpc6 −/− mice (Fig. 1C,D). Additionally, Trpc6 expression in kidneys after IRI was not altered compared to the sham kidneys ( Fig. 1E). At baseline Trpc6 −/− mice had similar blood parameters compared to WT mice (except higher but still normal concentrations of sodium and ionized Ca 2+ (Supplementary Table S2A). After IRI, WT and Trpc6 deficient mice developed similar hyperkalaemia and similar high serum levels of creatinine and urea nitrogen (Supplementary Table S2B). In contrast, concentrations of sodium and chloride were lower in Trpc6 −/− mice (Supplementary Table S2B).
The impact of Trpc6 deficiency on renal histopathology after IRI. To assess the degree of tubular damage in ischemic AKI kidney sections were analyzed by Periodic Acid-Schiff (PAS) staining ( Fig. 2A). IRI induced severe tubular damage and necrosis such as tubular epithelial swelling, loss of brush border, luminal dilatation with simplification of the epithelium, patchy loss of tubular epithelial cells with resultant gaps and exposure of denuded basement membrane, as well as obliterated tubular hyaline and/or granular casts ( Fig. 2A). Analysis of the kidney sections revealed no differences in tubular injury score and tubular necrosis score between Trpc6 −/− and WT mice (Fig. 2B,C).
The impact of Trpc6 deficiency on cellular infiltration and on the expression of calcium-binding proteins in the kidneys after IRI. Neutrophils infiltration after IRI-induced AKI contributes to inflammation and subsequent repair of injured kidneys 24,25 . Here, we determined neutrophils infiltration in renal IRI using immunofluorescence staining. Kidney sections were immunolabelled with the neutrophil marker Ly6B.2 26 . As shown in Fig. 3A, excessive Ly6B.2-positive neutrophils in renal interstitium were observed in mice underwent IRI (Fig. 3A). Trpc6 −/− mice showed similar amount of infiltratating Ly6B.2-positive cells after IRI as WT mice (Fig. 3B). Similarly, renal expression of neutrophil markers S100 calcium-binding protein A8 (S100a8) and S100 calcium-binding protein A9 (S100a9) were not different in IRI-damaged renal tissues of Trpc6 −/− and WT mice (Fig. 3C,D).

Expression of inflammatory markers in WT and Trpc6 −/− kidneys. Expression of inflammatory
molecules is low in the normal kidney but is markedly increased under pathophysiological conditions such as IRI-induced AKI 27 . By using qPCR, we examined the mRNA expression of cell adhesion molecules and inflammatory markers involved in renal IRI. IRI led to increased mRNA expression of all determined markers ( Fig. 4A-F), however Trpc6 −/− IRI kidneys displayed similar expression of interleukin 6 (Il6), tumor necrosis factor-alpha (Tnf-a), intercellular adhesion molecule 1 (Icam1), vascular cell adhesion molecule 1 (Vcam1) in comparison to WT IRI kidneys ( Fig. 4A-D). In addition, renal expression of chemokines such as chemokine (C-C motif) ligand 2 (Ccl2) and chemokine (C-C motif) ligand 2 receptor (Ccr2), were similar between Trpc6 −/− and WT mice (Fig. 4E,F).
The impact of Trpc6 pharmacological blockade on renal damage after renal IRI. To circumvent possible bias due to the genetical approach and to examine effects of pharmacological blockade of Trpc6 on renal damage, we used SH045 (a recently developed drug with high affinity and strong subtype selectivity toward TRPC6) in vivo in the ischemic AKI mouse model using two different ischemia time (milder with 17.5 min and www.nature.com/scientificreports/ a stronger with 20 min) (Fig. 5A). Renal concentrations of SH045 in both 30 min and 24 h after intravenous injection were similar between sham and IRI groups (Fig. 5B). Consistent with AKI data from Trpc6 −/− and WT mice, pharmacological blockade of TRPC6 had no effects on serum creatinine levels after 17.5 min or 20 min IRI-induced AKI in comparison to vehicle injected WT IRI mice (P = 0.23 and P = 0.76, respectively, Fig. 5C). In addition, renal mRNA expression of Ngal and Kim1 was similar in SH045-treated compared to vehicle-treated kidneys after IRI (Fig. 5D,E). Furthermore, SH045 did not affect blood parameters such as sodium, potassium, chloride, ionized calcium, carbon dioxide, glucose, urea nitrogen, hematocrit, hemoglobin, and the anion gap  The impact of Trpc6 pharmacological blockade on renal histopathology after IRI. Histological analyses and semi-quantitative scoring revealed no statistically significant differences in cortical tubular damage

The impact of Trpc6 pharmacological blockade on cellular infiltration and calcium-binding proteins on the kidneys after IRI.
Consistent with the results in AKI-induced Trpc6 −/− mice, SH045 treatment had no effect on renal infiltration of Ly6B.2-positive granulocytes after 17.5 min-or 20 min-IRI-induced AKI in comparison to vehicle-treated mice (Fig. 7A,B). In agreement with that, renal expression of S100a8 and S100a9 after 17.5 min-or 20 min-IRI is similar in mice treated with SH045 versus vehicle (Fig. 7C,D). Furthermore, similar results were also obtained in mice treated with BI-749327 compared to vehicle-treated mice (Supplementary Fig. S3G,H).
The impact of Trpc6 pharmacological blockade on expression of renal cytokines and chemokine after IRI. SH045-treated mice underwent 17.5 or 20 min IRI-induced AKI showed similar renal expression of Il6, Tnf-α, Icam1, Vcam1, Ccl2, and Ccr2 in comparison to vehicle-treated mice with the same IRI ( Fig. 8A-F).
In addition, the mRNA levels of these inflammatory markers in BI-749327-treated AKI mice were equivalent to vehicle-treated AKI mice ( Supplementary Fig. S3A-F).
In addition, activation of TRPC6 using hyperforin resulted in similar spontaneous decrease of perfusion pressure compared to control kidneys ( Supplementary Fig. S4A,D,E). Of note, there was also no difference in Angiotensin (Ang) II induced vasoconstrictions between kidneys perfused with SH045, BI-749327, or hyperforin versus control kidneys ( Supplementary Fig. S4F). Together, these results indicate no effect of TRPC6 modulation on renal arterial myogenic tone.

Discussion
In our previous study, we have shown that TRPC6 contributes to renal fibrosis and immune cell infiltration in a murine UUO model using Trpc6 −/− mice 9 . Renal IRI is a common cause of CKD. IRI is associated with calcium (Ca 2+ ) overload, ROS production, and immune responses which has been reported as crucial factors of tubular www.nature.com/scientificreports/ www.nature.com/scientificreports/ injury in IRI-induced AKI 12,15,16 . As a Ca 2+ -permeable cationic channel, TRPC6 can be activated by reactive oxygen species (ROS) and mediate podocyte injury in glomerular diseases 17,18 . In addition, TRPC6 is involved in immune responses regulating transendothelial migration, chemotaxis, phagocytosis, and cytokine release 19,20 . A recent study using pharmacological blockage of TRPC6 in mice demonstrated that decreased Ca 2+ entry due to TRPC6 contributed to reducing astrocytic apoptosis, cytotoxicity and inflammatory responses in cerebral ischemic/reperfusion insult 30 . Given that, in the present study we hypothesized that TRPC6 inhibition may also exhibit nephroprotective effects in AKI. To test this hypothesis, we applied the IRI experimental model on Trpc6 −/− mice and employed a pharmacological approach of in vivo TRPC6 inhibition by SH045 and BI-749327 in wild-type (WT) mice. www.nature.com/scientificreports/ In our study, Trpc6 −/− mice subjected to acute IRI showed no difference in renal function or tubular damage when compared to WT mice which underwent IRI. The Trpc6 −/− mouse model was established as a homozygous colony, therefore we used C57BL/6 J mice as control 9 . Given this limitation of our experimental approach and to account for possible confounding genomic and non-genomic effects of other TRPC channels caused by global loss of TRPC6 31 , we performed also studies using two different TRPC6 blockers SH045 and BI-749327 in C57BL/6 J WT mice to evaluate the role of TRPC6 in short-term AKI outcomes. Of note, TRPC6 and TRPC2, TRPC3, TRPC7 can form heteromeric channels with regulatory properties that are different from those of the homomeric TRPC channels 32,33 . Pharmacological inhibition allows high selectivity for inhibition of homomeric TRPC6 channels than for heteromeric channels in AKI, particularly compared to targeting TRPC6 using Trpc6 −/− animals. Thus, using the pharmacological approach can precisely allow investigating the specific effects of TRPC6 homomeric channels on AKI. We used SH045 and BI-749327, and performed studies with two different ischemic times in order to have a model for mild (17.5 min) and moderate (20 min) renal damage. These two periods of reversible ischemia were based on our earlier IRI studies with mice [34][35][36] . However, we still found no differences with respect to serum creatinine or tubular damage between mice undergoing IRI treated with TRPC6 blocker vs. vehicle in both models.
Previously, we demonstrated that inhibition of tubular epithelial NF-κB activity can ameliorate IRI-induced AKI in our experimental settings 36 . Thus, our experimental conditions enable to observe beneficial effects of therapeutic interventions, i.e. kidney injury potentially can be ameliorated in our AKI mouse model. According to the reported IC 50 -values for SH045 of 6 to 60 nM 37 and the pharmacokinetic analysis by non-compartment The semi-quantification renal neutrophils infiltration. (C) Renal mRNA levels of S100 calcium-binding protein A8 (S100a8). (D) Renal mRNA levels of S100 calcium-binding protein A9 (S100a9). Data expressed as means ± SEM (Sham n = 3 each, 17.5 min-IRI in vehicle and SH045 n = 5 each, 20 min-IRI in vehicle and SH045 n = 4 each, respectively). Two-way ANOVA followed by Sidak's multiple comparisons post hoc test. AU, arbitrary units. www.nature.com/scientificreports/ modeling 38 , SH045 can be considered as pharmacologically effective at tissue concentrations equal or higher than 22 ng/g. To confirm the drug is distributed in the kidney within the expected time, we measured the renal concentration of SH045 by using LC-MS/MS assay. As shown in Fig. 5B, the concentration of SH045 in renal tissue after 30 min post-injection (284.56 ng/g ± 144.76) was much higher than 22 ng/g. In case of BI-749327, Lin et al., reported that in concentrations of 10-30 mg/kg BI-749327 has renal protective effects in the UUO mouse model 14 . Therefore, the concentrations of pharmacological blockers used in this study were appropriate to inhibit the TRPC6 channels in vivo in our experiments.
It is now well established that inflammation plays an important role in AKI [39][40][41][42] . Shortly after endothelial or tubular epithelial cell damage, activation of resident renal inflammatory cells occurs. It leads to overproduction of cytokines, followed by recruitment and subsequent infiltration with different leukocytes subsets 43,44 . TRPC6 www.nature.com/scientificreports/ is expressed in a wide range of inflammatory and vascular cell types, including neutrophils, lymphocytes and endothelium 45 . During the acute phase response, TRPC6 plays a crucial role in neutrophil mobilization as it enhances chemotactic responses through increasing intracellular Ca 2+ concentration and promoting actin-based cytoskeleton remodeling 20,46 . Indeed, after renal IRI, we found a notable accumulation of neutrophils in the region of outer medulla. However, the infiltration of neutrophils in IRI Trpc6 −/− kidney was equivalent to IRI WT mice as well as in SH045-treated AKI mice compared to vehicle-treated AKI mice. Accordingly, inflammatory response including cytokines and chemokine overexpression was also similar in AKI mice with or without Trpc6 intervention. In addition, Trpc6 was normally expressed in kidney tissue 24 h after IRI. A recent study using single-cell RNA sequencing in clusters of inflammatory cells during AKI confirms a similar renal expression of Trpc6 mRNA in AKI versus control 47 . S100a8 and S100a9 are Ca 2+ binding proteins belonging to the S100 family, which are constitutively expressed in neutrophils and monocytes as a Ca 2+ sensor, participating in cytoskeleton rearrangement and arachidonic acid metabolism 48,49 . These two molecules modulate inflammatory response by stimulating recruitment of neutrophils and cytokine secretion 49 . We hypothesized that blocking intracellular influx of Ca 2+ in neutrophils through genetical or pharmacological TRPC6 inhibition might affect the S100a8/9mediated inflammation. However, we detected comparable renal expression of S100a8/9 after AKI in WT and Trpc6 −/− mice or in TRPC6 blockers-treated kidneys after AKI compared to respective control kidneys. Taken together, our data suggest no impact of TRPC6 to renal inflammatory reaction in the ischemic AKI. Of note, TRPC6 is also expressed in tubular epithelial cells and plays an important role in nephron physiology 50 . Although one study showed functional importance of TRPC6 in autophagy regulation in vitro using proximal tubular cells of Trpc6 deficient mice and TRPC6 overexpressing HK-2 cells 50 , our data argue against protective effects of TRPC6 in the tubular damage of AKI in vivo. After AKI, interstitial fibrosis formation is an important part of renal repair, yet excess leads to AKI-to-CKD transition. Wu et al., and our previous study showed that inhibition of TRPC6 function either by genetic deletion ameliorates renal interstitial fibrosis 10 . Besides, another study further demonstrated an obligate function for TRPC6 in promoting fibroblast transdifferentiation 51 . Therefore, inhibition of TRPC6 may have effects on renal fibrogenesis during AKI-to-CKD transition, and the beneficial effects of TRPC6 inhibition seen in the UUO model most likely involve fibroblast activation and transdifferentiation. Since its differentiation is an expected long-term outcome of AKI 52 , this might explain the lack of Trpc6 −/− effects on the short-term outcome of AKI in present study. In addition, renal microvascular dysfunction is considered playing critical role in acute kidney injury 53 . Due to unchanged myogenic tone after TRPC6 modulation in vascular smooth muscle cells using isolated perfused kidneys, it is unlikely that a vascular component represents a protective mechanism of renal TRPC6 inhibition.
In summary, the present study shows that TRPC6 inhibition has no effects on short-term outcome of AKI. Our results improve the understanding of the role of TRPC6 role in kidney diseases. Future studies should investigate if TRPC6 could have a protective role on long-term outcome of AKI. Our results may have clinical implications for safety and long-term outcome of humans with TRPC6 gene variations leading to familial forms of FSGS, with respect to the response of the kidney to acute ischemic stimuli.

Materials and methods
Animals. Male Trpc6 −/− mice (n = 10, homozygosity in a mix 129 Sv:C57BL/6J background) and wildtype (WT, C57BL/6J) control mice (n = 9) were used. Trpc6 −/− mice have been generated and characterized previously 9,31 . For the pharmacological study WT mice (C57BL/6J, Jackson Laboratory) were used. Mice were held in specific-pathogen-free (SPF) condition, in a 12:12-h light-dark cycle, and with free access to purified food (E15430-047, Ssniff, Soest, Germany) and water. Experiments were approved by the Berlin Animal Review Board, Berlin, Germany (No. G0178/18) and followed the restrictions in the Berlin State Office for Health and Social Affairs (LaGeSo) 54 . All experiments were performed in accordance with ARRIVE guidelines 55 .
Renal IRI model. Renal IRI was induced as described earlier 34 . Briefly, male mice (aged 14-18 weeks) were anaesthetized by isoflurane (2.3%) in air (350 ml/min). Preemptive analgesia with buprenorphine (0.2 mg/100 g) was used 56 . Mice were operated individually to ensure similar exposure to isoflurane 57 . Body temperature was maintained at 37 °C and monitored during surgery. Ischemia was induced after right-sided nephrectomy by clipping the pedicle of the left kidney for 17.5 or 20 min with a non-traumatic aneurysm clip (FE690K, Aesculap, Germany). Reperfusion was confirmed visually and the abdomen and the skin were sutured separately with a 5/0 braided-silk suture. After surgery mice had free access to water and chow. Body-warm sterile physiological saline solution (1 ml) was applied subcutaneously to every mouse. Sham operation was performed in a similar manner, except for clamping the renal pedicle. Twenty-four hours after reperfusion, mice were sacrificed by overdose of isoflurane and additionally cervical dislocation, and kidney and blood samples were collected for further analysis. The kidneys were divided into three portions. Upper pole of the kidney tissue was frozen and used for measurement of SH045 concentration. Middle part of kidney was immersed in 4% phosphate-buffered saline (PBS)-buffered formalin for histology, and the other left tissue was snap-frozen in liquid nitrogen for RNA preparation.
TRPC6 blocker. Larixyl-6-N-methylcarbamate, also called SH045, is a compound with high affinity and subtype selectivity toward TRPC6 described by Häfner et al. 22 . SH045 was initially dissolved in DMSO (ratio of DMSO to vehicle is 0.5%) and then in 5% Cremophor EL ® solution with 0.9% NaCl and used for intravenous injection (2 mg/kg) 30 min before IRI surgery in the pharmacological studies with WT mice. BI-749327 is an orally bioavailable TRPC6 antagonist as reported 14 . BI-749327 (MedChemExpress, New Jersey, USA) was dissolved in DMSO and then suspended in corn oil (final ratio of DMSO to corn oil is 5%). For administration, 30 mg/kg of the final dose was delivered to mice via oral gavage 60 min before IRI surgery. www.nature.com/scientificreports/ Blood measurements and drugs. To allow repeated blood measurements of sodium, potassium, chloride, ionized calcium, total carbon dioxide, glucose, urea nitrogen, creatinine, hematocrit, hemoglobin and anion gap within a short time interval in the same mouse, 95 μL blood was taken from the facial vein and parameters were measured using an i-STAT system with Chem8 + cartridges (Abbott, USA).
Quantitative real-time (qRT)-PCR. qRT-PCR was performed as described previously 9 . Briefly, Total RNA from snap-frozen kidneys were isolated using RNeasy RNA isolation kit (Qiagen, Australia) according to manufacturer's instruction after homogenization with a Precellys 24 homogenizator (Peqlab, Germany). RNA concentration and quality were determined by NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific, USA). Two micrograms of RNA were transcribed to cDNA (Applied Biosystems, USA). Quantitative analysis of target mRNA expression was performed with qRT-PCR using the relative standard curve method. TaqMan and SYBR green analysis was conducted using an Applied Biosystems 7500 Sequence Detector (Applied Biosystems, USA). The expression levels were normalized to 18S. Primer sequences are provided in Supplementary Table S1.
Histology and analysis. Formalin-fixed, paraffin-embedded sections (2 μm) of kidneys were subjected to Periodic acid Schiff (PAS) stain according to the manufacturer's protocols (Sigma, Germany). Semi-quantitative scoring of tubular damage was performed in a blinded manner in those 12 to 15 images at 200 × magnification per sample. Acute tubular injury (ATI) was observed in this study to assess the reversible tubular damage due to ischemia. The histologic features of ATI included one or more of the following lesions: tubular epithelial swelling with lucency of the cytoplasm, loss of brush border, luminal dilatation with simplification of the epithelium, and cytoplasmic vacuolization. Acute tubular necrosis (ATN) was also evaluated, which was indicated by patchy loss of tubular epithelial cells with resultant gaps and exposure of denuded basement membrane, evidence of cellular regeneration, as well as obliterated tubular hyaline and/or granular casts. The histological findings were graded from 0 to 3 according to the distribution of lesions: 0 = none; 1 = < 25%; 2 = 25-50%; 3 = > 50% 56 . The total score was calculated as sum of all morphological parameters. Tubular damage and tubular necrosis were quantified by an experienced renal pathologist who was unaware of Trpc6 genotypes or of treatment.

HPLC and tandem mass spectrometric method (LC-MS/MS). Concentration of SH045 in kidney
homogenates was measured as previously described 23 . In summary, analyses were performed with an Agilent 1260 Infinity quaternary HPLC system (Agilent Technologies, Germany) consisting of a G4225A degasser, G1312B binary pump, G1367E autosampler, G1330B thermostat, G1316A column oven and G4212B diode array detector, coupled to a tandem QTRAP 5500 hybrid linear ion-trap triple quadrupole mass spectrometer (AB SCIEX, Canada). Data were acquired and processed using Analyst software (Version 1.7.1, AB SCIEX). Linear regressions and calculations were done using Multiquant software (Version 2.1.1, AB SCIEX). Lower limit of quantification (LLOQ) and accuracy were determined for quantification of SH045 effects in kidney and both revealed as high as reported for plasma by Chai et al. 38 .
Immunofluorescence and analysis. We performed immunofluorescence similarly as previously described 34 . Two-μm thick sections of IRI-injured kidneys were post-fixed in ice-cold acetone, air-dried, rehydrated and blocked with 10% normal donkey serum (Jackson ImmunoResearch, America) for 30 min. Then sections were incubated in a humid chamber overnight at 4 °C with rat anti-Ly6B.2 (Gr1) (1:300; MCA771G; Bio-Rad AbD Serotec, Germany). The bound anti-Ly6B.2 antibody was visualized using Cy3-conjugated secondary antibody (1:500; Jackson ImmunoResearch, America) by incubating the sections for 1 h in a humid chamber at room temperature. Positive cells were counted in the outer medulla on ten non-overlapping view fields at 400 × magnification and mean cell numbers were taken for analysis.
Isolated perfused kidneys. Isolated kidneys were perfused in an organ chamber using a peristaltic pump (Instech, USA) at constant flow (0.3-1.9 ml/min) of oxygenated (95% O 2 and 5% CO 2 ) physiological salt solution (PSS) containing (in mmol/L) 119 NaCl, 4.7 KCl, 1.2 KH 2 PO 4 , 25 NaHCO 3 , 1.2 Mg 2 SO 4 , 11.1 glucose, 1.6 CaCl 2 ). Hyperforin (Sigma-Aldrich, USA), BI-749327, and SH045 were dissolved in DMSO. Before application, the aliquots were dissolved 1:1000 in PSS. So, the final concentration of DMSO did not exceed 0.1%. The concentrations of hyperforin, BI-749327, SH045 were 10 µM, 100 nM, and 100 nM, respectively. We used PSS as a solvent for Ang II (LKT Laboratories Inc., USA) and applied it at 10 nM concentration. Drugs were added to the perfusate. Perfusion pressure was detected using pressure transducer after an equilibration period of 60 min. Powerlab system (AD Instruments, Colorado Springs) was used for data acquisition and analysis. Ang II-induced pressor effects were normalized to the maximal pressor effect obtained with KCl (60 mmol/L) 58  Statistics. Statistical analysis was performed using GraphPad 5.04 software. Study groups were analyzed by one-way ANOVA using Turkey's post-hoc test or two-way ANOVA using Sidak's multiple comparisons post hoc test. Data are presented as mean ± SEM. P values < 0.05 were considered as statistically significant.
Experimental statement. All methods were carried out in accordance with relevant guidelines and regulations.